Complete bibliographic citations of the references referred to herein by number in parentheses can be found in the Bibliography section, immediately preceding the claims.
This invention relates to compositions and methods useful for the prevention, treatment and early diagnosis of Lyme disease in humans and other animals. More particularly, this invention relates to outer surface protein (Osp) polypeptides which are able to elicit in a patient the formation of a specific immune response which is effective to diagnose, predict successful eradication of infection or protect against Lyme disease in a mammalian host. This invention also relates to a screening method to detect anti-Osp borreliacidal antibody activity, and antibodies which react with a protein fragment encoded by a DraI-SmaI DNA fragment of OspC. Also within the scope of this invention are antibodies directed against Osp polypeptides, diagnostic kits comprising the antibodies or the polypeptides, and vaccines using borreliacidal epitopes of OspA, OspB or OspC or a conserved DNA sequence fragment together with or without a vaccine carrier.
Lyme disease (Lyme borreliosis) is spread by a bite from an infected tick, and is the most commonly reported tick-borne infection in Europe and North America (1). This multi-system disorder has caused significant morbidity worldwide.
Lyme disease is caused by the spirochete Borrelia burgdorferi (B.b.), which is primarily transmitted during the blood feeding of Ixodes ssp ticks. Initially, spirochetes infect the skin and, in the majority of early cases, cause an erythema migrans lesion (2). Those stricken may not become ill for weeks, and nervous system symptoms (headaches, dizziness, hearing problems, tingling and trouble concentrating) may not occur for weeks or months. It is now known that infection can spread to the nervous system or joints, and the risk of neurological or joint complications increases the longer the disease goes untreated. Infection can be asymptomatic, or have a range of clinical presentations, depending on the tissues affected, the duration of infection, host factors such as the vulnerability of the immune system and immunogenetic factors which could predispose a patient to the development of certain complications.
The treatment of symptomatic patients is currently with a range of antibiotics, e.g., tetracyclines, penicillin and cephalosporins, but studies show mixed results. Left untreated, the bacteria can spread to the central nervous system, heart, brain, or joints, causing arthritis, cardiac infections and neurologic problems, and in rare cases, death (3-6).
Upon infection with borrelia, B cells in the body begin producing antibodies which recognize the foreign organism. There are at least two functional types of antibodies produced in response to a borrelia infection. One response is a nonspecific binding/opsonizing (coating) response which xe2x80x9cmarksxe2x80x9d the antigen and may result in ingestion of B.b. by phagocytic cells. These non-specific antibodies are produced against proteins common among several bacterial species (viz. 41 kDa proteins to many bacterial flagella). Thus, these antibodies will recognize and attach to similar antigens on other bacteria. Because of this, diagnostic tests which detect these non-specific binding/opsonizing antibodies are generally nonspecific.
A second functional antibody response is the production of borreliacidal (lethal) antibodies which specifically recognize epitopes on some individual proteins of the B.b. organisms. After attachment of these antibodies to the B.b. organisms, complement interacts with the antibodies to form a membrane attack complex which kills the B.b. organism without the necessity of scavenging by phagocytic cells. This highly specific borreliacidal antibody response is often detectable within the first 2 weeks of infection. The successful detection and induction of borreliacidal antibodies is gaining importance in the Lyme disease diagnostic and prevention armamentarium.
Shortly after the discovery of Lyme borreliosis, researchers determined that vaccination of experimental animals with whole B.b. provided protection against challenge (7,8). Additional studies established the role of antibody-mediated protection and confirmed the ability of vaccination with B.b. to induce antibodies which provide protection against B.b. infection (9-11). To date, vaccination of animals with Osps of B.b., especially OspA (12-14), OspB (12,14), and OspC (14-16), have provided protection against infection with the Lyme spirochete. Protection after vaccination with OspA and OspB have been shown to be due to the induction of borreliacidal antibodies which specifically killed the B.b. organisms (13,14,17-24). In contrast, anti-OspC borreliacidal antibodies have not been detected after vaccination (14-16) and investigators have postulated that protection after vaccination with OspC is due to other mechanisms (16).
Most of the efforts to date have focused on developing an OspA vaccine primarily because of the large amounts of OspA expressed on the surface of many B.b. laboratory isolates. To date, most borrelia spirochetes have had outer surfaces comprised mainly of OspA. Therefore, it has been believed that inducing borreliacidal antibodies against OspA would provide protection against the spirochetes. SmithKline Beecham (Philadelphia, Pa.) and Pasteur Merieux Connaught (Lyon, France) have developed vaccines based on the generation of borreliacidal antibodies to OspA. The SmithKline Beecham vaccine has been approved for general use, and the Pasteur Merieux Connaught vaccine is currently being assessed by the U.S. Food and Drug Administration. As one would expect, OspA vaccines have been shown to be effective in animal models when the animals have been needle challenged. In addition, OspA vaccination has provided protection against ticks infected with B.b. However, protection against a tick challenge has been dependent on the presence of high levels of anti-OspA borreliacidal antibodies. Schwan et al. (28) recently demonstrated that spirochetes in infected ticks downregulate OspA on their surface during ingestion of a blood meal. Thus, OspA vaccines must induce high titers of anti-OspA borreliacidal antibodies to destroy the spirochetes in the midgut of infected ticks before they downregulate OspA. Therefore, the duration of high titers of anti-OspA borreliacidal antibodies is a critical determinant of the long-term efficacy of an OspA vaccine.
The applicants recently demonstrated the inability of a commercial OspA vaccination to sustain adequate levels of anti-OspA borreliacidal antibodies in humans (23). It is also unlikely that an anamnestic response will occur quickly enough to eliminate B.b. organisms from infected ticks. In support, infection with B.b. has been documented in OspA-vaccinated humans and dogs (26,27). These results highlight the need to evaluate other Lyme borreliosis vaccine components.
In addition, Lyme disease is ordinarily diagnosed by detecting antibodies in the blood or cerebral spinal fluid, but the most commonly used tests are often inaccurate. False-negative, and more commonly, false-positive results continue to plague the serodiagnosis of Lyme disease. Several schemes using conventional diagnostic assays have been developed to more accurately detect Lyme disease. Unfortunately, little improvement has occurred and misdiagnosis continues to cause significant economic and health effects. In addition, the recent approval of an OspA Lyme disease vaccine will further confound conventional diagnostic testing. Thus, a sensitive and specific Lyme disease test which can be made widely available as a commercial kit and can discriminate between vaccinated individuals and patients with Lyme disease is still needed.
Detection of borreliacidal antibodies may also solve this problem. Borreliacidal antibodies have been shown to serve as the basis for a sensitive and highly specific serodiagnostic test (17,25-27). In fact, a diagnostic assay for Lyme disease, which detects this antibody response, has been previously developed, patented (37), and is commercially available. This test relies on detection of highly specific borreliacidal antibodies which are induced by several B.b. Osps shortly after infection. It is important to note if sufficiently high levels of borreliacidal antibodies are induced by vaccination, the vaccinee is protected. However, if an individual is infected before borreliacidal antibodies are present, the person can get Lyme disease despite the eventual presence of high concentrations of borreliacidal antibodies. This test provides a more sensitive and specific alternative approach for confirming Lyme disease. In addition, antibody detected by the borreliacidal antibody test does not correlate with antibody detected by conventional assays. In hamster studies, detection of borreliacidal antibodies decreased with elimination of B.b. from the host (43). In contrast, the antibody responses detected by conventional assays remain elevated or continues to expand. These results suggest that the borreliacidal antibody test is a prognostic indicator for clearance of the spirochete.
Callister et al. (30) recently showed the ability to increase the sensitivity of the borreliacidal antibody test while maintaining the exquisite specificity through the use of a test antigen (B.b. 50772) which does not contain OspA or OspB on the surface. The increased sensitivity with B.b. 50772 is proposed by the applicants to be due to detection of borreliacidal antibodies against OspC or other Osps. These results greatly increase the utility of the borreliacidal antibody testing procedure disclosed in U.S. Pat. No. 5,385,826 to Schell et al., which is incorporated herein in its entirety.
In addition, several investigators have demonstrated the ability of vaccination with OspC to protect laboratory animals against needle challenge (14-15) and natural infection (16). A recent investigation also showed that passive transfer of immune sera to OspC could resolve arthritis, carditis and infection with B.b. (44). These results demonstrate that vaccination with OspC may be more effective than vaccinations with OspA. However, since high concentrations of anti-OspC borreliacidal antibodies have not been detected in immune serum (14-16), there has been speculation that additional mechanisms are responsible for OspC-mediated protection (16). Not knowing the mechanism makes it much more difficult to pursue an OspC Lyme disease vaccine. In addition, the pursuit of OspC as a Lyme borreliosis vaccine candidate has been hindered because OspC appears to be more immunologically and genetically heterogeneous than OspA (35-37), causing researchers to speculate that development of a comprehensive OspC vaccine is economically unfeasible. However, Schwan et al. (28) recently showed that relatively large amounts of OspC are rapidly synthesized by B.b. shortly after attachment of infected ticks to mammalian hosts and that OspA is no longer expressed in high concentration on the surface of B.b.
It has recently been reported that B.b. organisms upregulate OspC and concomitantly downregulate OspA shortly before tick inoculation of the host with the spirochete (31,32). This explains why anti-OspC antibodies are among the first antibody responses detected in patients with early Lyme borreliosis. In response, several investigators have attempted or are attempting to develop enzyme-linked immunosorbent assays (ELISA) using whole recombinant OspC proteins (38-42). These diagnostic assays have been reasonably sensitive but have continued to lack specificity. In addition, the anti-OspC antibody responses detected by these assays may remain elevated or continue to expand even after clearance of B.b. from the host. Of particular concern is the propensity of OspC to cross-react with antibodies in sera from patients with other illnesses such as cytomegalovirus (CMV) or Epstein-Barr virus (EBV) and give a false-positive reaction. Because of this significant lack of specificity and the lack of prognostic potential, OspC ELISAs remain significantly less than ideal.
The present invention is directed to an isolated, immunogenic polypeptide fragment of OspC of Borrelia burgdorferi consisting essentially of an epitope of OspC having an amino acid sequence as shown in residues 145 to 194 of SEQ. ID. NO: 2.
The present invention is also directed to an isolated polypeptide having an amino acid sequence as shown in residues 145 to 194 of SEQ. ID. NO: 2.
The present invention is also directed to an isolated DNA molecule encoding a polypeptide having an amino acid sequence as shown in residues 145 to 194 of SEQ. ID. NO: 2.
The present invention is also directed to an expression vector comprising an isolated DNA encoding a polypeptide having an amino acid sequence as shown in residues 145 to 194 of SEQ. ID. NO: 2.
Further, the present invention is directed to a pharmaceutical composition to vaccinate against and to treat borrelia infection in mammals, including humans, the composition comprising an amount of an isolated polypeptide consisting essentially of an amino acid sequence as shown in residues 145 to 194 of SEQ. ID. NO: 2, the amount being effective to prevent or to treat borrelia infection in mammals.
The present invention is also directed to a method to prevent and to treat borrelia infection in mammals, including humans, comprising administering to a patient in need thereof an amount of an isolated polypeptide consisting essentially of an amino acid sequence as shown in residues 145 to 194 of SEQ. ID. NO: 2, the amount being effective to prevent or to treat borrelia infection in the patient.
The present invention is also directed to a kit for diagnosing borrelia infection in mammals, including humans, the kit comprising an isolated polypeptide having an amino acid sequence as shown in residues 145 to 194 of SEQ. ID. NO: 2, disposed in a suitable container therefor, and instructions for use of the kit.
The present invention is also directed to a method to detect borrelia infection in mammals, including humans, comprising contacting a body fluid of a mammalian host suspected to suffer from borrelia infection with an isolated polypeptide having an amino acid sequence as shown in residues 145 to 194 of SEQ. ID. NO: 2; and then determining whether the isolated polypeptide is conjugated to antibodies present in the body fluid of the mammalian host, whereby presence of conjugation indicates presence of borrelia infection in the host.
Advantageously, the polypeptide of this invention is not confounded by previous vaccinations against Lyme disease. Detection of anti-OspC borreliacidal antibodies against the Dra fragment is useful for diagnosing Lyme disease in patients from anywhere in the world and also for vaccination against Lyme disease caused by Borrelia ssp. There are 3 major species of Lyme disease spirochetes now known: B.b., B. garinii and B. afzelii. The inventors have detected anti-OspC borreliacidal antibodies in patients from Slovenia infected with B. afzelii. They detected these anti-OspC borreliacidal antibodies by using B.b. 50772, the same isolate used to detect a response in patients infected with B.b. Thus, the Dra fragment of OspC appears to be conserved in all species of borrelia.
A Dra fragment-based ELISA is an excellent complementary test to the borreliacidal antibody test. Most importantly, a Dra fragment-based ELISA is easily manufactured as a commercial kit and discriminates between patients vaccinated with early Lyme disease.
A test which detects borreliacidal antibodies against the borreliacidal epitope(s) of OspA and OspB in addition to OspC would give reduced cross-reactivity, increased specificity, greater accuracy, and fewer false-positive diagnoses. Detection of anti-OspC borreliacidal antibodies advantageously gives an early diagnosis which anti-OspA and anti-OspB borreliacidal antibodies cannot do. However, the identification of the borreliacidal epitope(s) of OspB and OspA are also valuable additions to a diagnostic test.
Inclusion of borreliacidal epitopes of OspA and OspB with the Dra fragment of OspC also yields a more comprehensive Lyme borreliosis vaccine.
Further objects and advantages of the invention will appear more fully from the following detailed description of the preferred. embodiment of the invention made in conjunction with the accompanying tables and drawings.